The objective of this research program is to elucidate structure- function relationships of selected carbon-carbon lyases. These experiments depend upon use of an analog of substrate which is an alkylating agent. This reagent binds to the active site in a catalytic step and turns over as does substrate. Inactivation results from the alkylation of a protein-bound basic group which has geometry allowing its interaction with the bound substrate analog. Kinetic methodology has been devised which can distinguish a system where both catalysis and alkylation occur at the same protein site. In addition, reduction of inactivated enzyme produces a secondary amine via azomethine reduction. Thus the reagent is a bridging agent which crosslinks the active site between two functional groups implicated in catalysis. It is thus possible, through the use of substrate analogs and appropriate enzymes, to study active site geometry. The model system for these studies, developed by the applicant, is the bromopyruvate inactivation of 2-keto- 3-deoxy-6-P-gluconate aldolase in which it has been demonstrated by the above approach that the gamma-carboxylate of a glutamate occurs adjacent to the Schiff's base-forming lysine in active site tertiary structure. It is now possible, through the use of radioactive reagent, to generate peptides whose sequence determination will contribute to a three dimensional model of the active site of this C-C lyase. Further studies are being undertaken with a lyase synthesizing the C-4 anomer of KDPG, viz. KDPGal aldolase. These studies will allow one to compare the active site three dimensional structure of enzyme classes which handle substrates of opposite configuration. This technique is also being used to study N- acetylneurimanic acid aldolase. In addition, metal dependent aldolases will be investigated in order to gain insight into active site tertiary structure of these aldolases. Such knowledge of active site tertiary structure is crucial to the intelligent design of drugs which will either cure or moderate metabolic disease.